ABSTRACT Idiopathic pulmonary fibrosis (IPF) is chronic, progressive lung disorder with high mortality, and limited therapeutic options. Studies performed during Cycle I of this translational PPG (tPPG) have uncovered a critical role for redox imbalance in activated myofibroblasts (myo-Fbs) that may drive disease progression in IPF. In animal models of lung fibrosis, targeting the reactive oxygen species (ROS)-generating enzyme, NADPH oxidase-4 (NOX4), by genetic approaches or by a pharmacologic approach (GKT137831, to be tested in Phase IIb clinical trial, see Project 1) protects from fibrosis. Recent studies by UAB-tPPG investigators have discovered a metabolic reprogramming of myo-Fbs characterized by elevated aerobic glycolysis (aGLY), and mitochondrial dysfunction. Our data demonstrate that the pro-fibrotic cytokine, transforming growth factor-?1 (TGF-?1), induces generation of tricarboxylic acid (TCA) cycle metabolites, including succinate and fumarate, which are known to stabilize/activate hypoxia-inducible factor 1? (HIF-1?) (7-9). Our preliminary studies show that TGF-?1-induced activation of HIF-1? is inhibited in Fbs with genetic deletion in NOX4 (NOX4-/-). Additionally, we have established a novel 3D-spheroid tissue culture system that provides a complementary approach to test for myo-Fb invasiveness and screen for anti-fibrotic compounds in a patient-specific manner. Paracrine effects of activated macrophages (see Project 3) and B-lymphocytes (see Project 4) are critical in regulating Fb and myo-Fbs phenotypes; however, the roles of NOX enzymes and potential for metabolic reprogramming of myo-Fbs by these cell types are not well understood. The hypothesis to be tested in this project is that NOX4 mediates fibrogenic effects by metabolic reprogramming that involves mitochondrial dysfunction and generation of TCA cycle metabolites to confer an invasive and apoptosis-resistant phenotype to myo-Fbs, thus, impeding fibrosis resolution. The specific aims to be tested are: (1) to determine whether the NOX4 expression and/or the invasive capacity of Fbs is predictive of severity and/or progression of IPF; and to characterize the heterogeneity in responses to anti- fibrotic drugs; (2) to determine the mechanisms by which NOX4 metabolically reprograms myo-Fbs to induce apoptosis resistance and invasion; and determine whether these pro-fibrotic myo-Fb phenotypes are modulated by activated macrophages and/or B-cells; and (3) to determine whether the protective effect of genetic/pharmacologic NOX4 inhibition in animal models of fibrosis is mediated by reversal of pro-fibrotic metabolic programs. These completion of these studies will: (a) link the biology of NOX4 with metabolic reprogramming; (b) provide new insights into the role of intermediary metabolism in determining fibrotic gene expression and pro- fibrotic cellular phenotypes; and (c) utilize novel 3D-spheroid tissue culture models to study cell invasion, to phenotype multi-cellular disease processes, and to assess responsiveness to specific drug therapies.